Luna SARS-CoV-2 RT-qPCR Multiplex Assay Protocol (NEB #E3019)

1. Thaw Luna Probe One-Step RT-qPCR 4X Mix with UDG, SARS-CoV-2 Primer Mix (10X), Positive Control and Nuclease-free Water at room temperature.
   
   
2. Once completely thawed, mix each component by inversion, pipetting or gentle vortexing. Centrifuge briefly to collect liquid to the bottom of the tube and then place in a cold rack at 4°C or ice.
   
   
3. Detemine the total number of reactions per assay run. Each assay run should include the following:
   
   
   1. One Positive Control using the SARS-CoV-2 Positive Control provided in the kit, as template
      
      
   2. One Negative Control using Nuclease-free Water provided in the kit, as template
      
      
   3. t number of Test Samples
      
      

   The total number of reactions n = 1 (Positive Control) + 1 (Negative Control) + t (Test Samples)

   An overview of the reaction setup for each reaction type is described in the table below.

   COMPONENT	VOLUME PER 20 µl REACTION
   	TEST SAMPLES	POSITIVE CONTROL	NEGATIVE CONTROL
   Luna Probe One-Step RT-qPCR 4X Mix with UDG	5 µl	5 µl	5 µl
   SARS-CoV-2 Primer/Probe Mix (N1/N2/RP) (10X)	2 µl	2 µl	2 µl
   Test Sample	2–10 µl	–	–
   SARS-CoV-2 Positive Control (N gene)	–	2 µl	–
   Nuclease-free Water	to 20 µl	11 µl	13 µl

4. Combine the components indicated below on ice for the number of reactions (n) being evaluated. Volumes include 10% overage to accommodate transfer loss from pipetting. The recommended input volume (v) is 2-10 μl for Test Samples and 2 μl for the Positive Control (as indicated below, water will be used to make up any difference in volume between the PC and the Test Samples).

   SARS-CoV-2 RT-qPCR Assay Mix:

   COMPONENT	VOLUME FOR ONE REACTION	VOLUME FOR n REACTIONS
   Luna Probe One-Step RT-qPCR Mix with UDG	5 µl	5 µl x n x 1.1
   SARS-CoV-2 Primer/Probe Mix (N1/N2/RP) (10X)	2 µl	2 µl x n x 1.1
   Nuclease-free Water	(13- u) µl	(13- u) µl x n x 1.1

5. Mix thoroughly but gently by pipetting or vortexing, centrifuge briefly to collect liquid to the bottom of the tube.
   
   
6. Aliquot assay mix (20- v) μl into qPCR tubes or wells. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles. If the input volume for the Test Sample is greater than 2 μl, add (v -2) μl nuclease-free water to the Positive Control tube or well.
   
   For example: If n = 50 and the Test Sample volume is 5 μl, add 15 μl of the assay mix into 50 tubes or wells. Then add an additional 3 μl of water into the Positive Control tube or well.
   
   

7. Add v μl Test Sample, v μl nuclease-free water as Negative Control, and 2 μl Positive Control, to the appropriate qPCR tubes or wells on the plate. Mix thoroughly but gently by pipetting up and down, taking care to avoid bubbles.

   Example sample layout for a 96-well plate:
   
   
   
   

8. Seal tubes with flat, optically transparent caps; or seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.
   
   
9. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
   
   
10. Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.

Programming the Real-time PCR Instrument

Use the scanning mode that will detect signal in all channels on the real-time instrument.
For faster results, the “Fast” ramp speed mode can be used where available (e.g., QuantStudio®, 7500 Fast instruments).

Target fluorophore assignment:

Data Analysis

• For basic information regarding data analysis on specific real-time PCR instruments, please refer to the user manual of the respective instrument.

• After the run is complete, inspect the amplification plot to ensure that the baseline threshold was set within the PCR exponential phase and above any background signal.